For a number of reasons including ease of cell culture, genetic manipulation and experimental design the social amoeba Dictyostelium discoideum has long been a model system for investigating the morphological and molecular events of chemotaxis and development. Starvation of Dictyostelium initiates a 24-h developmental process which begins with the pulsed secretion of cAMP by a fraction of the amoebae towards which neighboring amoebae chemotax. Interaction of the secreted cAMP with the G-protein-coupled cAMP receptor (cAR1) on the plasma membranes of neighboring cells initiates a series of molecular and morphological events including enhanced expression of cAR1 and adenylyl cyclase A (ACA), cell elongation and polarization and chemotaxis. Release of G from the heterotrimeric G-protein coupled to cAR1 activates myosin II. G also activates two synergistic and partially redundant RasC- and RasG-signaling pathways. One pathway activates target of rapomycin complex 2 (TORC2) and protein kinase B (PKB) initiating polymerization of actin at the front of the cell, which, together with contraction of actomyosin II at the rear, supports chemotaxis towards the aggregation centers. A second Ras pathway activates phosphatidylinositol 3-kinase (PI3K) at the cells leading edge, which catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) to which cytoplasmic regulator of adenylyl cyclase (CRAC) binds and activates membrane-associated ACA. PIP3 also contributes to the TORC2 pathway which induces actin polymerization. TORC2 contributes to activation of ACA and, independently of G, binding of cAMP to cAR1 leads to phosphorylation and activation of extracellular signal regulated kinase 2 (ERK2) which increases cAMP concentration by inhibiting its hydrolysis by a phosphodiesterase. ACA-containing vesicles translocate to the rear of chemotaxing cells where secretion of cAMP creates a cell-to-cell cAMP signal relay resulting in head-to-tail streams of cells that aggregate into tight mounds of 100,000 or more cells in about 12 h. Over the next 12 h, the multi-cellular mounds differentiate through several morphological stages developing into mature fruiting bodies comprising a spore head supported by a stalk. In an appropriate nutritional environment, spores germinate into amoebae and the life-cycle begins anew. Last year we reported that ectopic expression of Y53A-actin inhibits cell streaming (although individual cells chemotax normally) and blocks development beyond the mound stage. The developmental phenotype of Y53A-actin cells correlates with an inhibition of intracellular and intercellular cAMP-signaling pathways, most significantly the trafficking of ACA-vesicles to, and secretion of cAMP at, the rear of chemotaxing cells (Shu et al., 2010). It is highly likely that the underlying cause of these phenomena is the disorganized actin cytoskeleton of amoebae expressing Y53A-actin. Whereas WT-cell cytoskeletons comprise a mostly homogeneous array of long filaments, cytoskeletons of Y53A-actin-cells contain many short filaments and numerous bundles and aggregates of short and long filaments similar to the structures formed by copolymerization of Y53A-actin and WT-actin in vitro. Interestingly, a developmental phenotype similar to that of Dictyostelium amoebae expressing Y53A-actin, i.e. inhibition of both aggregation streams and development of mounds to mature fruiting bodies, had previously been described for Polysphondylium (a close relative of Dictyostelium) upon deletion of the actin crosslinking protein cortexillin I (Fey and Cox, Dev. Biol.212, 414-424, 1999). The molecular events underlying this phenotype were not explored, as we have now done for Dictyostelium cortexilllin (ctx)-null cells. Dictyostelium ctxI and ctxII, 444 and 441 amino acids, respectively, are parallel dimers with a coiled coil domain and two globular heads which contain actin-binding sites. Cortexillin I also has a putative PIP2-binding site at its C-terminus and a second, and stronger, actin-bundling domain in the C-terminal region that is inhibited by PIP2. Both cortexillins accumulate in the cortex of vegetative cells and the cortical region of spreading cells where, together with myosin II, they bundle and cross-link actin filaments in an anti-parallel fashion. In motile cells, both cortexillins are enriched at the leading edge and, to a lesser extent, at the rear. Cortexillins also localize to the cleavage furrow of dividing cells independently of myosin II where, together with myosin II, they increase cleavage furrow stiffness. We now find that both head-to-tail cell streaming of Dictyostelium amoebae into multi-cellular mounds and development of the mounds to mature fruiting bodies are partially inhibited in ctxA-- and ctxB- cells (ctxA and ctxB are the genes coding for proteins cortexillin I and II, respectively), and completely inhibited in ctxA-/B- -cells, as they are in cells expressing Y53A-actin. The double deletion of cortexillin I and II alters the actin cytoskeleton, with thick bundles of filaments in the cell cortex, and substantially inhibits all molecular responses to extracellular cAMP. Synthesis of cAR1 and ACA is inhibited and activation of ACA, RasC and RasG, phosphorylation of ERK2, activation of TORC2 and stimulation of actin polymerization and myosin assembly are all greatly reduced. As a consequence, cell streaming and development are completely blocked. On the other hand, translocation of ACA-vesicles from the front to the rear of the cell is not significantly affected in the cortexillin-knockout cells. Expression of ACA-YFP in the ctxI/ctxII-null cells significantly rescues the wild-type phenotype, but expression of cAR1-YFP dies not. Thus, whereas impairment of cell streaming and development of Y53A-actin cells may be caused primarily by inhibition of ACA-vesicle translocation to, and secretion of cAMP at, the rear of the cell (Shu et al., 2010), inhibition of cell streaming and development of ctxA-/B- cells probably results principally from decreased secretion of cAMP due to inhibition of ACA synthesis. The phenotypes of Y53A-cells and ctxA-/B- cells demonstrate the critical importance of a properly organized actin cytoskeleton for cAMP-induced signaling pathways.